1. Field of the Invention
The present invention relates to a novel member of the serine protease inhibitor (serpin) superfamily of proteins, in particular the plasminogen activator inhibitor (PAI) protein family. More specifically, isolated nucleic acid molecules are provided encoding the pancreas-derived plasminogen activator inhibitor (PAPAI) protein. Plasminogen activator inhibitor polypeptides are also provided. The present invention further relates to methods for treating physiologic and pathologic disease conditions and diagnostic methods for detecting pathologic disorders.
2. Related Art
The mammalian serine protease inhibitors (serpins) are a superfamily of single chain proteins that contain a conserved structure of approximately 370 amino acids and generally range between 40 and 60 kDa in molecular mass. .alpha..sub.1 -Antitrypsin (also known as .alpha..sub.1 -proteinase inhibitor) is a characteristic member of the serpin family in that it is a single chain glycoprotein of nearly 400 amino acid residues that functions by forming a tight 1:1 complex with its cognate protease, neutrophil (leucocyte) elastase, which subsequently slowly dissociates to yield active enzyme and inactive cleaved inhibitor (Carrell, R. W. et al., Cold Spring Harbor Symposia on Quantitative Biology 52:527-535 (1987)). The reactive center of the serpins is typically formed by an X-Ser that acts as a substrate for the target serine protease: .alpha..sub.1 -antitrypsin has a Met-Ser reactive center with the methionine residue providing a putative cleavage site for neutrophil elastase.
The majority of serpins function as protease inhibitors and so are involved in regulation of several proteinase-activated physiological processes, such as blood coagulation, fibrinolysis, complement activation, extracellular matrix turnover, cell migration and prohormone activation (Potempa, J. et al., J. Biol. Chem. 269:15957-19560 (1994)). As noted, serpins inhibit proteolytic events by forming a 1:1 stoichiometric complex with the active site of their cognate proteinases, which is resistant to denaturants (Cohen, A. B. et al., Biochemistry 17:392-400 (1987). The serpins include, but are not limited to, .alpha..sub.1 -antitrypsin (.alpha..sub.1 -proteinase inhibitor), antithrombin III, plasminogen activator inhibitor 1 (PAI-1), plasminogen activator inhibitor 2 (PAI-2), .alpha..sub.1 -antichymotrypsin, and .alpha..sub.2 -antiplasmin (Huber, R. and Carrell, R. W., Biochemistry 28:8951-8966 (1989).
The plasminogen activator system is responsible for the degradation of intravascular blood clots, and also contributes to extracellular proteolysis in a wide variety of physiological processes of normal development and pathological processes in the etiology of diseases such as tumor invasion and metastasis (Andreasen, P.-A., et al., Int. J. Cancer 72(1):1-22 (1997); Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997)). Plasmin, a trypsin-like protease, is generated from its precursor plasminogen by the action of plasminogen activators, of which there are two types: tissue-type plasminogen activator (also known as tissue plasminogen activator) and urokinase. Plasmin degrades fibrin and several extracellular matrix and adhesion proteins and activates procollagenases.
Plasminogen activation is a highly regulated process. Precise, coordinated, spatial and temporal regulation is afforded by the interaction of a variety of mechanisms. These mechanisms include (1) inhibition by specific plasmin and plasminogen activator inhibitors; (2) binding of plasminogen, plasminogen activators, and inhibitors to fibrin, extracellular matrix proteins, and specific cell surface receptors; (3) release of tissue plasminogen activator and inhibitors from intracellular storage granules; (4) regulation of gene expression of plasminogen activators and inhibitors; (5) an autocrine feedback loop whereby plasmin-mediated activation of latent forms of growth factors regulates the expression of activators and inhibitors; and (6) clearance of free and inhibitor-bound activators via receptors (Bachmann, F. et al., Fibrinolysis, in: Thrombosis Haemostasis 1987, Verstraete, M. et al., eds., Leuven University Press (1987); Dan.o slashed., K. et al., Adv. Cancer Res. 44:139 (1985); Pollanen, J. et al., Adv. Cancer Res. 57:273 (1991); Vassalli, J. D. et al., J. Clin. Invest. 88:1067 (1991); Carmeliet, P. et al., Thromb. Haemost. 74:429 (1995); Andreasen, P. A. et al., Mol. Cell. Endocrinol. 68:1 (1990); Loskutoff, D. J., Fibrinolysis 5:197 (1991); Keski-Oja, J. et al., Semin. Thromb. Hemost. 17:231 (1991); Blasi, F., BioEssays 15:105 (1993); Andreasen, P. A. et al., FEBS Lett. 338:239 (1994); Bu, G. et al., Blood 83:3427 (1994); and Camani, C. et al., Int. J. Hematol. 60:97 (1994)).
Strong clinical and experimental evidences have suggested a causal role for the tumor-associated urokinase-type PA (u-PA) and the receptor u-PAR in cancer invasion and metastasis (Andreasen, P.-A., et al., Int. J. Cancer 72(1):1-22 (1997); Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997); Duggan, C., et al., Br. J. Cancer 76(5):622-627 (1997)). Consistent with its role in cancer metastasis, overexpression and unrestrained activity of u-PA has been shown to be a prognostic marker in many different types of human cancer (Schmitt, M., et al., Fibrinolysis 6(Suppl. 4):3-26 (1992); Schmitt, M., et al., J. Obstet. Gynaecol. 21:151-165 (1995); Brunner, N., et al., Cancer Treat. Res. 71:299-309 (1994); Kuhn, W., et al., Gynecol. Oncol. 55:401-409 (1994); Ganesh, S., et al., Cancer Res. 54:4065-4071 (1994); Nekarda, H., et al., Cancer Res. 54:2900-2907 (1994); Duffy, M.-J., J. Clin. Cancer Res. 2:613-618 (1996)). The down-regulation of u-PA may occur at the levels of transcriptional regulation of the genes and through interaction with specific endogenous inhibitors such as plasminogen activator inhibitor (PAI).
Only two plasminogen activator inhibitors are known. These are plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2, respectively). PAI-1 and PAI-2 regulate mitogenesis, adhesion of myeloid cells, fusion of myoblasts, and migration of endothelial cells (Fazioli, F. et al., Trends Pharm. Sci. 15:25-29 (1995)). Indeed, PAI-1 and PAI-2 are involved in many physiological and pathological processes, including normal pregnancy, preeclampsia, intrauterine growth retardation, wound healing, tumor cell invasion and metastasis, inflammation and arthritis, inflammatory bowel disease, appendicitis, complications from systemic lupus erythematosus, ovulation and prostatic involution and osteonecrosis (Kruithof, E. K. O. et al., Blood 86:4007 (1995)).
Both PAI-1 and PAI-2 have been shown to inhibit extracellular matrix degradation in vitro (Cajot, J.-F., et al., Proc. Natl. Acad Sci. USA 87:6939-6943 (1990); Baker, M.-S., et al., Cancer Res. 50:4676-4684 (1990)). These results suggest that the inhibitory activity of PAls might be important in inhibiting tumor malignant progression leading to metastasis. In fact, administration of a recombinant PAI-2 to mice decreases tumor growth (Astedt, B., et al, Fibrinol. 9:175-177 (1995)), whereas overexpression of either PAI-1 or PAI-2 inhibits tumor metastasis (Muller, B., et al., Proc. Natl. Acad Sci. USA 92:205-209 (1995); Soff, G.-A., et al., J. Clin. Invest. 96:2593-2600 (1995)).
In breast cancer, it has been reported that uPA and PAI-1 are statistically independent, strong prognostic factors for disease free and overall survival, i.e., high tumor levels are associated with a poor prognosis and are conductive to tumor cell spread and metastasis (Brunner, N., et al., Cancer Treat. Res. 71:299-309 (1994); Duffy, M., et al., Cancer 62:531-533 (1988); Duggan, C., et al., Int. J. Cancer 61:597-600 (1995); Schmitt, M., et al., Br. J. Cancer 76(3):306-311 (1997)). Immunohistochemical staining has detected PAI-I expression at stromal fibroblasts surrounding tumor nodules or at tumor margins (Bianchi, E., et al., Int. J. Cancer 60:597-603 (1995)). The production of PAI-I by the tumor stroma may represent a host defensive response to the excessive proteolysis. In contrast to PAI-I, high level PAI-2 expression may be a favorable prognostic marker in breast cancer (Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997); Duggan, C., et al., Br. J. Cancer 76(5):622-627 (1997)). In breast carcinomas with high uPA values, PAI-2 was associated with a prolonged relapse-free survival, metastasis-free survival, and overall survival (Bouchet, C., et al., Br. J Cancer 69:398-405 (1994)). In relation to the clinicopathological findings, an inverse correlation between PAI-2 mRNA expression and lymph node metastasis was reported in breast cancers (Sumiyoshi, K., et al., S. Int. J. Cancer 50:345-348 (1992)). In this study, the expression of uPA and PAI-1 was significantly correlated with negative expression of PAI-2; and a low level of PAI-2 expression was significantly associated with lymph node involvement (Sumiyoshi, K., et al., Int. J. Cancer 50:345-348 (1992)). PAI-2 expression is detected predominantly in malignant mammary epithelial cells of primary carcinomas but is also present in stromal cells (Andreasen, P.-A., et al., Int. J. Cancer 72(1):1-22 (1997)). These results indicate that PAI-2 may play a critical role in inhibition of extracellular matrix degradation mediated by plasminogen activator during tumor cell invasion and metastasis.
In view of the wide range of roles that plasminogen activator inhibitors play in physiologic and pathologic processes, there is a continuing need for the isolation and characterization of novel plasminogen activator inhibitors.